We will investigate the 3D structure, catalytic mechanism, and regulation of soluble methane monooxygenase (MMO). MMO initiates the oxidation of CH4 to CO2 by methanotrophic bacteria. In this way, the atmospheric egress of nearly all of the enormous quantity of CH4 (greenhouse gas with 20 times the potency of CO2) generated by anaerobic bacteria is prevented. MMO also adventitiously catalyzes the oxidation of many other chemicals fostering applications in synthesis as well as biodegradation of abundant pollutants with human toxicity (e.g. trichloroethylene). MMO from Methylosinus trichosporium OB3b is composed of 3 proteins: hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB). MMOH has a bis-u-hydroxo-bridged dinuclear Fe cluster needed for catalysis. Spectroscopic studies (optical, EPR, Mossbauer, EXAFS, ENDOR, rRaman, fluorescence, NMR, MCD, and CD), turnover of diagnostic substrates, and transient kinetics are being used to study the structure and mechanism. Transient kinetic studies have revealed 2 stable and 7 transient intermediates in the reaction cycle. 1 intermediate, compound Q, contains a bis-u-oxo-Fe(IV)-Fe(IV) cluster which reacts directly with CH4 to give CH3OH. Q is the first intermediate isolated in an oxygenase that can attack unactivated hydrocarbons. Ongoing studies suggest that MMOR and MMOB regulate catalysis by increasing the rate of Q formation and by controlling the rate of substrate entry into the active site of MMOH based on size. MMOB mutants have been purified that allow the rate of each step in the catalytic cycle to be individually regulated. Our recent studies have defined the interaction surfaces between the MMO components. The proposed studies will utilize fluorescence energy transfer, cross-linking, mass spec, and crystallography techniques to define the spatial orientation of the components as well as conformational changes that gate substrate binding. Reaction cycle intermediates will be trapped using new approaches based on steady state stabilization and surface freeze quenching. These will be spectroscopically characterized by newly developed X-ray absorption and cryoreduction techniques. This work should give us insight into: 1) novel O2 activation chemistry, 2) the nature of Q, 3) a new regulation strategy, and 4) design of small molecule catalysts for hydrocarbon oxidation. Finally, lessons learned from MMO should apply to the structurally and mechanistically similar human ribonucleotide reductase, which generates the building blocks for DNA.